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Nat Protoc. 2013 May;8(5):935-48. doi: 10.1038/nprot.2013.048. Epub 2013 Apr 18.

Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores.

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  • 1Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut, USA.

Abstract

This protocol describes an assay that uses suspended nanomembranes called nanodiscs to analyze fusion events. A nanodisc is a lipid bilayer wrapped by membrane scaffold proteins. Fluorescent lipids and a protein that is part of a fusion machinery, VAMP2 in the example detailed herein, are included in the nanodiscs. Upon fusion of a nanodisc with a nonfluorescent liposome containing cognate proteins (for instance, the VAMP2 cognate syntaxin1/SNAP-25 complex), the fluorescent lipids are dispersed in the liposome and the increase in fluorescence, initially quenched in the nanodisc, is monitored on a plate reader. Because the scaffold proteins restrain pore expansion, the fusion pore eventually reseals. A reducing agent, such as dithionite, which can quench the fluorescence of accessible lipids, can then be used to determine the number of fusion events. A fluorescence-based approach can also be used to monitor the release of encapsulated cargo. From data on the total cargo release and the number of the much faster lipid-mixing events, the researcher may determine the amount of cargo released per fusion event. This assay requires 3 d for preparation and 4 h for data acquisition and analysis.

PMID:
23598444
[PubMed - indexed for MEDLINE]
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